Makiko Negishi

Entrapping desired amounts of actin filaments and molecular motor proteins in giant liposomes.

We have successfully prepared cell-sized giant liposomes encapsulating desired amounts of actoHMM, a mixture of actin filament (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), in the presence of 5 mM MgCl 2 and 50 mM KCl. We employed a spontaneous transfer method to prepare those liposomes. In the absence of HMM, F-actin was distributed homogeneously inside the liposomes. In contrast, when F-actin was encapsulated in liposomes together with HMM, network structures were generated. Such network structures are attributable to the cross-linking of F-actin by HMM.

Transformation of ActoHMM Assembly Confined in Cell-Sized Liposome

To construct a simple model of a cellular system equipped with motor proteins, cell-sized giant liposomes encapsulating various amounts of actoHMM, the complexes of actin filaments (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), with a depletion reagent to mimic the crowding effect of inside of living cell, were prepared. We adapted the methodology of the spontaneous transfer of water-in-oil (W/O) droplets through a phospholipid monolayer into the bulk aqueous phase and successfully prepared stable giant liposomes encapsulating the solution with a physiological salt concentration containing the desired concentrations of actoHMM, which had been almost impossible to obtain using currently adapted methodologies such as natural swelling and electro-formation on an electrode. We then examined the effect of ATP on the cytoskeleton components confined in those cell-sized liposomes, because ATP is known to drive the sliding motion for actoHMM. We added α-hemolysin, a bacterial membrane pore-forming toxin, to the bathing solution and obtained liposomes with the protein pores embedded on the bilayer membrane to allow the transfer of ATP inside the liposomes. We show that, by the ATP supply, the actoHMM bundles inside the liposomes exhibit specific changes in spatial distribution, caused by the active sliding between F-actin and HMM. Interestingly, all F-actins localized around the inner periphery of liposomes smaller than a critical size, whereas in the bulk solution and also in larger liposomes, the actin bundles formed aster-like structures under the same conditions.

Protein Synthesis in Giant Liposomes Using the In Vitro Translation System of Thermococcus kodakaraensis

An in vitro translation system, based on cell components of the hyperthermophilic archaeon, Thermococcus kodakaraensis, has previously been developed. The system has been optimized and applied for protein production at high temperatures (60-65°C). In this paper, we have examined the possibilities to utilize this system at a lower temperature range using green fluorescence protein (GFP) as the reporter protein. By optimizing the composition of the reaction mixture, and adding chaperonins from the mesophilic Escherichia coli, the yield of protein production at 40°C was increased by fivefold. For liposome encapsulation of the optimized system, water-in-oil cell-sized emulsions were prepared by adding the translation system/GFP mRNA mixture to mineral oil supplemented with 1,2-dioleoyl-sn -glycero-3-phosphatidylcholine (DOPC). Giant liposomes were formed when these emulsions passed across a water/oil interface occupied with DOPC. The liposomes were incubated at 40°C for 90 min, and fluorescence was examined by laser confocal microscopy. A significant increase in average fluorescence intensity was observed in liposomes with GFP mRNA, but not in those without mRNA. Our results indicate that the T. kodakaraensis in vitro translation system is applicable for protein production within giant liposomes, and these artificial cell models should provide the methodology to reconstitute various cell functions from a constitutional biology approach.

Construction of Cell-Sized Liposomes Encapsulating Actin and Actin-Cross-linking Proteins

To shed light on the mechanism underlying the active morphogenesis of living cells in relation to the organization of internal cytoskeletal networks, the development of new methodologies to construct artificial cell models is crucial. Here, we describe the successful construction of cell-sized liposomes entrapping cytoskeletal proteins. We discuss experimental protocols to prepare giant liposomes encapsulating desired amounts of actin and cross-linking proteins including molecular motor proteins, such as fascin, alpha-actinin, filamin, myosin-I isolated from brush border (BBMI), and heavy meromyosin (HMM). Subfragment 1 (S-1) is also studied in comparison to HMM, where S-1 and HMM are single-headed and double-headed derivatives of conventional myosin (myosin-II), respectively. In the absence of cross-linking proteins, actin filaments (F-actin) are distributed homogeneously without any order within the liposomes. In contrast, when actin is encapsulated together with an actin-cross-linking protein, mesh structures emerge that are similar to those in living motile cells. Optical microscopic observations on the active morphological changes of the liposomes are reported.

How Does the Mobility of Phospholipid Molecules at a Water/Oil Interface Reflect the Viscosity of the Surrounding Oil?

The mobility of phospholipid molecules at a water/oil interface on cell-sized phospholipid-coated microdroplets was investigated through the measurement of diffusion constants by fluorescence recovery after photobleaching. It is found that the diffusion constant of phospholipids showed the relation D approximately (eta water + eta oil) -0.85, where D is the diffusion constant, eta water is the viscosity of water, and eta oil is the viscosity of oil. This observation indicates that the viscosity of the surrounding oil is the primary factor that determines the diffusibility of phospholipids at a water/oil interface.

Specific Transformation of Assembly with Actin Filaments and Molecular Motors in a Cell-Sized Self-Emerged Liposome

Eukaryotes, by the same combination of cytoskeleton and molecular motor, for example actin filament and myosin, can generate a variety of movements. For this diversity, the organization of biological machineries caused by the confinement and/or crowding effects of internal living cells, may play very important roles.

Reconstruction of motile actin networks in giant liposome

To construct a simple model cellular system exhibiting the property of self-propelled motion, cell-sized giant liposomes encapsulating desired amounts of actoHMM, a mixture of actin filament (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), have been prepared. We adapted the methodology of spontaneous transfer of a water droplet through oil/water interface in the presence of phospholipid and successful obtained stable giant liposome with the inner physiological biopolymer solution. We introduced ATP to the bathing solution of liposome encapsulating actoHMM, in which bilayer membrane α-hemolysin, a bacterial membrane pore-forming toxin, is embedded. In this system, ATP is supplied into the inner volume of liposome through the protein pores in a passive manner. Accompanied by the ATP supply, actin networks or bundles that have encapsulated in the liposomes exhibited specific morphological change, being attributable to the active sliding between F-actin and HMM. Remarkable difference in the behavior of F-actins is found; i.e., inside the liposome, almost all the F-actins situate around the inner periphery of the liposome, whereas, in the bulk solution, actin bundles form an aster-like structure.

Real-world modeling of artificial motile cell

Using a spontaneous transfer method, cell-sized giant liposomes encapsulating desired amounts of actoHMM, a mixture of actin filament (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), have been successfully constructed in the presence of 5 mM MgCl2 and 50 mM KCl. The encapsulated actoHMM formed self-organized actin network-like structures, and non-spherical liposomes were obtained in a reproducible manner. In order to power the system with actoHMM in an effective manner, we have tried to supply ATP through protein pores embedded in the closed bilayer membrane. By the ATP supply, the network structures of F-actin formed inside the liposomes showed redistributions, which are attributable to the sliding between F-actin and HMM. This study serves as the first step in developing motile giant liposomes containing actoHMM, and in generating spontaneous motion in a system similar manner as in living cells.

Emergence of Superstructures from a Homogeneous Lipid Sphere

The spontaneous generation of a periodic hexagonal superstructure on a giant phospholipid sphere (GPS) with a diameter of 20-200 microm was studied. The GPS was composed of ternary phospholipids consisting of dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), and dioleoylphosphatidylinositol-bisphosphate (DOPIP(2)). GPSs were prepared by natural swelling of a lipid film formed on a glass substrate. A GPS with a homogeneous lipid mixture tends to form a two-layered structure between the surface and inner parts; the surface layer is attributed to a DOPIP(2) rich region (we call this layer SL), and the interior is rich in DOPE and DOPC (we call this layer IL). A hexagonal superstructure develops in the SL, and the topology then changes to form multiple-doughnut structures. Finally, myelin-like tubes are generated through symmetry breaking of the doughnutlike structures. The time-dependent change in the surface-area expansion of a GPS is shown to obey the logistic growth model, and this is attributed to the kinetic process of phase segregation between the surface and bulk phase of the GPS.

Radius-dependent phase behavior: Giant DNA and alginate in a cell sized sphere

We study the phase behavior when a mixture of two semi-flexible polymers, giant DNA and alginate, is highly confined mixture in a cell-sized scale space. The characteristic lengths of giant DNA is different from those of alginate. Through the microscopic observation, we found that the volume/surface area ration and the higher-order structure of giant semi-flexible polymer are the crucial determinants of the phase behavior in cell-sized scale space. When the volume/surface area ration is ≤ 1.0 under the osmotic pressure which is caused by the smaller polymer is small, the larger polymer can be depleted on the surface. When the volume/surface area ration is ≤ 1.0 with the higher osmotic pressure which is caused by the smaller polymer, the depletion of the larger polymer on the surface causes the phase-separation between two polymers within the space. When the volume/surface area ration is > 1.0 with higher osmotic pressure, the elongation force of the meta-stable coil becomes the dominant factor to decide the phase behavior in a cell-sized scale space.